CN112430254A - Anticoagulant active peptide derivative and preparation method and application thereof - Google Patents
Anticoagulant active peptide derivative and preparation method and application thereof Download PDFInfo
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- CN112430254A CN112430254A CN202011335865.5A CN202011335865A CN112430254A CN 112430254 A CN112430254 A CN 112430254A CN 202011335865 A CN202011335865 A CN 202011335865A CN 112430254 A CN112430254 A CN 112430254A
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- active peptide
- anticoagulant
- anticoagulant active
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/02—Linear peptides containing at least one abnormal peptide link
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention discloses an anticoagulant active peptide derivative and a preparation method and application thereof, and relates to the field of biological medicines. The amino acid sequence of the anticoagulant active peptide derivative is as follows: X-Phe-Lys-His-Met-Asn-GLu. Wherein the terminal group X is p-hydroxycinnamic acid, 2-amino-3- (2, 3-dihydroxyphenyl) propionic acid and 2-amino-3- (4-hydroxy-3-methoxyphenyl) propionic acid. The anticoagulant active peptide derivative prepared by the invention has high anticoagulant and thrombolytic activities, long half-life period and high stability; and has strong antioxidant activity.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to an anticoagulant active peptide derivative, and a preparation method and application thereof.
Background
The thromboembolic disease is called as the hidden killer of cardiovascular and cerebrovascular diseases, it can affect all organs and systems of the whole body, about 3 per thousand people generate different forms of thrombotic diseases every year, the number of people who die from cardiovascular and cerebrovascular diseases in China per year reaches more than 300 million, and the number of death caused by the hidden killer accounts for 51 percent of the total death number of the whole world every year. 75% of the surviving patients are disabled, and more than 40% of the patients are disabled seriously, which threatens human health.
Thrombus refers to a semi-clot formed on the surface of a blood vessel or the endocardium of a heart by blood components during the blood flow. Thrombotic diseases include arterial thrombosis, venous thrombosis, microthrombosis, thromboembolic diseases, and the like. Normally, coagulation and anticoagulation are in a dynamic balance in vivo, and when abnormal coagulation forms thrombus, anticoagulation medicine is used for inhibiting coagulation and thrombolytic medicine is used for enhancing fibrinolysis system effect, which are measures for correcting imbalance in vivo after disease occurs. In fact, diseases are caused by abnormal coagulation state in vivo and final thrombosis, and relatively long pathological processes exist in the process. By obtaining the information of the occurrence and the development of the disease in the monitoring of the index change of the blood coagulation state of the patient, the formation of thrombus can be prevented at an early stage, and the guide and the evaluation can be made on the treatment prognosis of the patient who has suffered from the thrombus.
At present, the main anticoagulant drugs clinically used at home and abroad are common heparin, low molecular heparin, warfarin and the like, and the drugs have the defects of side effects of thrombocytopenia, hemorrhage and the like or slow effect taking and the like. The polypeptide anticoagulant drug has the outstanding advantages of quick response, low side effect and the like, and is a key development direction of anticoagulant drugs in the future. Pharmacokinetic studies indicate that polypeptide/protein drugs are cleared in vivo mainly through degradation, excretion, and receptor-mediated endocytosis. Wherein the polypeptide factor with the molecular weight less than 20kDa is easily filtered by glomerulus in the metabolic process; the polypeptide factor is partially degraded by protease in the polypeptide factor and excreted from urine when passing through the renal tubule, and thus has a short half-life. Therefore, the development of long-acting polypeptide/protein drugs has become an important direction for the secondary development of the first generation of genetically engineered polypeptide/protein drugs.
Disclosure of Invention
The invention aims to provide an anticoagulant active peptide derivative, a preparation method and application thereof, wherein the anticoagulant active peptide derivative has high anticoagulant and thrombolytic activities, long half-life period and high stability; and has strong antioxidant activity.
The technical scheme adopted by the invention for realizing the purpose is as follows:
an anticoagulant active peptide, the amino acid sequence of which is: Phe-Lys-His-Met-Asn-GLu. The anticoagulant polypeptide prepared by the invention has higher anticoagulant activity and can be used for making up for the defects of heparin medicines; meanwhile, the prepared active peptide can directly permeate gastric digestive juice to enter small intestine, so that the activity of the polypeptide is better protected, and the high anticoagulation effect is exerted; and can effectively dissolve thrombus, and when the concentration reaches 1mg/mL or more, the thrombus dissolving effect is obvious. Compared with natural active peptide, the artificially synthesized polypeptide has longer half-life period and better stability.
Preferably, the anticoagulant active peptide has anticoagulant and thrombolytic effects.
Preferably, the anticoagulant activity of the anticoagulant active peptide is > 150 ATU/mL.
The invention also aims to provide application of the anticoagulant active peptide in preparing medicines for treating thrombotic cardiovascular and cerebrovascular diseases.
The invention also aims to provide application of the anticoagulant active peptide in preparing anticoagulant products.
The invention also discloses functional food of anticoagulant active peptide in preparation of anticoagulant and thrombolytic effects.
Preferably, the functional food is a powder, a tablet, an oral liquid or a capsule.
An anticoagulant active peptide derivative, the sequence of which is: X-Phe-Lys-His-Met-Asn-GLu, wherein the terminal group X is p-hydroxycinnamic acid or 2-amino-3- (2, 3-dihydroxyphenyl) propionic acid or 2-amino-3- (4-hydroxy-3-methoxyphenyl) propionic acid.
The anticoagulant active peptide is modified by p-hydroxycinnamic acid or 2-amino-3- (2, 3-dihydroxyphenyl) propionic acid or 2-amino-3- (4-hydroxy-3-methoxyphenyl) propionic acid to prepare the polypeptide derivative, so that the anticoagulant activity and thrombolytic performance are further enhanced, the half-life period is higher, and the stability in vivo is improved. In addition, the prepared polypeptide derivative has enhanced antioxidant performance.
Preferably, in the sequence, Phe is in the D configuration or L configuration, and the remainder are in the L configuration.
Preferably, the terminal group X has the formula:
or2-amino-3- (2, 3-dihydroxyphenyl) propionic acid; or2-amino-3- (4-hydroxy-3-methoxyphenyl) propionic acid.
A preparation method of anticoagulant active peptide derivatives is synthesized by adopting a known FMOC-short peptide solid phase synthesis method, and specifically comprises the following steps:
s1: swelling the resin;
s2: carrying out suction filtration on the first amino acid, removing a solvent through a sand core, adding 2-3 times of mol of Fmoc-amino acid, adding 2-3 times of mol of DIEA, 2-3 times of mol of HBTU, finally adding a small amount of DMF for dissolving, oscillating for 1-2 h, and alternately cleaning for 6 times by using DMF and DCM, wherein DMF (10mL/g) is twice, methanol (10mL/g) is twice, and DMF (10mL/g) is twice;
s3: deprotection, adding 15-20 mL of 20% piperidine DMF solution (10mL/g), pumping out the piperidine solution after 10-15 min, adding 10-15 mL of 20% piperidine DMF solution (10mL/g), and pumping out the piperidine solution after 15-20 min; detecting, namely taking dozens of resins, washing the resins with ethanol for three times, adding ninhydrin, KCN and a phenol solution, respectively one drop, heating the mixture at 105-110 ℃ for 5min, and turning dark blue to be a positive reaction;
s4: according to the operation principle of S2-S3, when the second and third amino acids are connected in succession, the second and third amino acid synthesis raw materials are sequentially added, and are sequentially connected from right to left to obtain amino acid sequences connected from left to right;
s5: connecting an X group, leaching out a solvent through a sand core, adding 2-3 times of mol of p-hydroxycinnamic acid, adding 2-3 times of mol of DIEA and 2-3 times of mol of HBTU, finally adding a small amount of DMF for dissolving, oscillating for 1-1.5 h, and alternately cleaning for 6 times by using DMF and DCM;
s6: cleavage of active peptide derivatives from the resin to prepare cleavage solutions: TFA (trifluoroacetic acid) 94.5%, EDT 2.5% and TIS 2.5%; and (3) putting the resin into a flask or a centrifuge tube, shaking the resin and the cutting fluid at a constant temperature for 120min according to the ratio of 10-11.5 mL/g. Performing suction filtration, collecting filtrate, adding diethyl ether, performing chromatography with diethyl ether to obtain polypeptide derivatives, filtering, washing the filter cake with diethyl ether for six times, volatilizing the filter cake at normal temperature, and blowing the residual diethyl ether in the filter cake with nitrogen to obtain crude anticoagulant bioactive peptide derivatives;
s7: and (3) purifying the anticoagulant active peptide derivative crude product by using HPLC.
Preferably, the sequence of the anticoagulant active peptide is: X-Phe-Lys-His-Met-Asn-GLu, wherein Phe is in D configuration or L configuration, and the rest is in L configuration; the specific structural formula is as follows:
alternatively, the first and second electrodes may be,
the invention also discloses the application of the anticoagulant active peptide derivative in preparing medicines for treating thrombotic cardiovascular and cerebrovascular diseases.
The invention discloses functional food of anticoagulant active peptide derivatives in preparation of anticoagulant and thrombolytic effects, wherein the functional food is powder, tablets, oral liquid or capsules.
Compared with the prior art, the invention has the following beneficial effects:
the anticoagulant polypeptide synthesized by the invention has higher anticoagulant activity and thrombolytic performance, and can be used for making up the defects of heparin drugs; meanwhile, the prepared active peptide can directly permeate gastric digestive juice to enter small intestine, so that the activity of the polypeptide is better protected, and the high anticoagulation effect is exerted; compared with natural active peptide, the peptide has longer half-life period and better stability. The peptide derivative obtained by chemically modifying the active peptide by adopting p-hydroxycinnamic acid or 2-amino-3- (2, 3-dihydroxyphenyl) propionic acid or 2-amino-3- (4-hydroxy-3-methoxyphenyl) propionic acid has stronger anticoagulation activity and thrombolytic performance, longer half-life period and improved in-vivo stability; meanwhile, the prepared polypeptide derivative has higher antioxidant performance.
Therefore, the invention provides an anticoagulant active peptide derivative, a preparation method and application thereof, wherein the anticoagulant active peptide derivative has high anticoagulant and thrombolytic activities, long half-life period and high stability; and has strong antioxidant activity.
Drawings
FIG. 1 shows the results of an anticoagulation activity test in test example 1 of the present invention;
FIG. 2 shows the results of the thrombolytic effect test in test example 2 of the present invention;
FIG. 3 shows the results of the antioxidant activity test in example 4 of the present invention.
Detailed Description
The technical solution of the present invention is further described in detail below with reference to the following detailed description and the accompanying drawings:
example 1:
1. raw materials and related reagents for synthesis:
Fmoc-L-GLu (ODmab) -OH, Eimei technologies, Inc.;
Fmoc-L-Asn (Trt) -OH, Suzhou Tianma pharmaceutical group;
Fmoc-L-Met(O2) -OH, gill biochemical (shanghai) ltd;
Fmoc-L-His (Boc) -OH, Gill Biochemical (Shanghai) Co., Ltd;
Fmoc-L-Lys (Boc) -OH, Gill Biochemical (Shanghai) Co., Ltd;
Fmoc-L-Phe-OH, Gill Biochemical (Shanghai) Ltd;
p-hydroxycinnamic acid, Shanghai Ji to Biochemical technology, Inc.
2. The preparation of the anticoagulant active peptide derivative is synthesized by adopting an FMOC-short peptide solid-phase synthesis method, and specifically comprises the following steps:
s1: weighing 0.8g AM Resin with substitution degree of 0.4mmoL/g in a solid phase synthesizer, adding 10mL anhydrous dichloromethane (hereinafter represented by DCM), placing on a shaking table, and shaking for 30min to fully swell the Resin;
s2: taking the first amino acid, filtering the solvent by a sand core, adding 3 times mol of Fmoc-L-GLu amino acid, adding 3 times mol of DIEA, 3 times mol of HBTU, finally adding a small amount of DMF for dissolving, oscillating for 1.5h, and alternately washing 6 times by DMF and DCM, namely twice DMF (10mL/g), twice methanol (10mL/g) and twice DMF (10 mL/g);
s3: deprotection, adding 15mL of 20% piperidine DMF solution (10mL/g), draining the piperidine solution after 10min, adding 10mL of 20% piperidine DMF solution (10mL/g), and draining the piperidine solution after 20 min; detecting, namely taking dozens of resins, washing the resins with ethanol for three times, adding ninhydrin, KCN and a phenol solution, respectively one drop, heating the mixture at 105-110 ℃ for 5min, and turning dark blue to be a positive reaction;
s4: according to the operating principle of S2-S3, when a second amino acid and a third amino acid are sequentially added, the second amino acid and the third amino acid are sequentially added, and the amino acid synthetic raw materials are sequentially connected from right to left, and the obtained amino acid sequence connected from left to right is Phe-Lys-His-Met-Asn-GLu;
s5: connecting X groups, filtering off a solvent through a sand core, adding 3 times of mol of p-hydroxycinnamic acid, adding 3 times of mol of DIEA and 3 times of mol of HBTU, finally adding a small amount of DMF to dissolve, oscillating for 1h, and alternately cleaning for 6 times by using DMF and DCM;
s6: cleavage of active peptide derivatives from the resin to prepare cleavage solutions: TFA (trifluoroacetic acid) 94.5%, EDT 2.5% and TIS 2.5%; and (3) putting the resin into a flask or a centrifuge tube, shaking the resin and the cutting fluid at a constant temperature for 120min according to the proportion of 10 mL/g. Performing suction filtration, collecting filtrate, adding diethyl ether, performing chromatography with diethyl ether to obtain polypeptide derivatives, filtering, washing the filter cake with diethyl ether for six times, volatilizing the filter cake at normal temperature, and blowing the residual diethyl ether in the filter cake with nitrogen to obtain crude anticoagulant bioactive peptide derivatives;
s7: and (3) taking the crude anticoagulant active peptide derivative for purification by HPLC:
putting 200mg of the anticoagulant active peptide derivative crude product into a vessel, dissolving the anticoagulant active peptide derivative crude product by using 4mL of 50% acetonitrile aqueous solution, and performing ultrasonic treatment for 2 min; filtering the solution with 0.45um filter membrane; analysis conditions were as follows: taking 3 mu L of the crude anticoagulant active peptide derivative solution, wherein the mobile phase is water and acetonitrile (the initial ratio is 90 percent of water and 10 percent of acetonitrile, the final ratio is 10 percent of water and 90 percent of acetonitrile), the time is 50min, the flow rate is2.5 mL/min, gradient elution is carried out, HPLC is balanced for 5min by using the initial gradient first, and then sample injection is carried out; the sample from the detector is collected. And performing conventional purity identification on the prepared sample, wherein the purity reaches 98.3%. And (3) carrying out freeze-drying on the purified solution under conventional conditions to obtain the anticoagulant active peptide derivative.
The structural formula of the anticoagulant active peptide derivative obtained in the embodiment is as follows:
example 2:
an anticoagulant active peptide derivative has the following structural formula:
the preparation process of the anticoagulant active peptide derivative is the same as that in the embodiment 1, and the purity of the anticoagulant active peptide derivative is 96.4%.
Example 3:
an anticoagulant active peptide derivative has the following structural formula:
the preparation process of the anticoagulant active peptide derivative is the same as that in the embodiment 1, and the purity of the anticoagulant active peptide derivative is 96.9%.
Example 4:
an anticoagulant active peptide derivative has the following structural formula:
the preparation process of the anticoagulant active peptide derivative is the same as that in the embodiment 1, and the purity of the anticoagulant active peptide derivative is 97.4%.
Example 5:
the preparation process of an anticoagulant active peptide derivative is the same as that in example 1, and the purity of the anticoagulant active peptide derivative is 95.8%.
Example 6:
an anticoagulant active peptide derivative has the following structural formula:
the preparation process of the anticoagulant active peptide derivative is the same as that in the embodiment 1, and the purity of the anticoagulant active peptide derivative is 97.8%.
Comparative example 1:
preparation of an anticoagulant active peptide: based on the prior art, the active peptide, Phe-Lys-His-Met-Asn-GLu, is extracted from minced fillet. The method specifically comprises the following steps:
degreasing, namely adding the minced fillet into the mixture in a volume ratio of 1: 1, degreasing with a mixed solution of n-hexane and absolute ethyl alcohol, extracting at 50 ℃ for 4h, repeating for 2 times, filtering, naturally drying and crushing the obtained filter cake to obtain degreased minced fillet;
and (3) performing enzymolysis, namely taking the degreased minced fillet according to a material-liquid ratio of 1: adding 10g/mL of water, uniformly mixing, heating at 90 ℃ for 15min, adjusting the pH of the solution to 8.0 by using 0.5M HCl, then adding chymotrypsin-trypsin (the addition amount is 8000u/mL), stirring at 40 ℃ and 360rpm for enzymolysis for 2h, heating at 100 ℃ for enzyme deactivation for 10min, centrifuging at 12000rpm for 10min, and taking the supernatant to obtain an enzymolysis solution; measuring the activity of antithrombin;
separating and purifying, namely performing ultrafiltration, ion exchange and gel filtration chromatography on the enzymolysis solution by using a 1.0kDa ultrafiltration membrane, and then performing reversed phase High Performance Liquid Chromatography (HPLC) purification to obtain the anticoagulant active peptide.
Comparative example 2:
the preparation of an anticoagulant active peptide differs from example 1 in that: not linked to the X group.
Test example 1:
determination of antithrombin Activity of samples by Thrombin titration improvement
(1) Preparing thrombin solutions with different concentrations
0.05moL/L Tris-HCl solution containing 0.05moL/L NaCL, pH7.4, was prepared to prepare 0.5% fibrinogen. Preparing thrombin solutions with different concentration gradients: 20NIH/mL (as a baseline concentration, i.e., 1 fold), 5 μ L of the solution was a titration volume, i.e., 1V, of 0.1 ATU:
20NIH ÷ (1000 μ L ÷ 5 μ L) ═ 20NIH ÷ 200 ═ 0.1NIH, and since 1NIH ═ 1ATU, 0.1ATU ═ 0.1NIH, 20NIH/mL (as a reference concentration, i.e., 1 time), 40NIH/mL (i.e., 2 times), 100NIH/mL (i.e., 5 times), and the like.
(2) Determination of anticoagulant Activity of samples
Several small test tubes of 7.5mm × 100mm were taken, 200 μ L of 0.5% fibrinogen was added, 0.5mg of the sample was added, and the mixture was placed in a 37 ℃ constant temperature water bath for 5 min.
Firstly, 5 mu L of thrombin solution which is 5 times of that of one part of the raw materials is dripped into one part of the raw materials, the raw materials are quickly shaken up, and if the raw materials are solidified within 1min, the anticoagulant activity contained in 50 mu L of hydrolysis extract is less than 0.5 ATU. In order to accurately know the anticoagulant activity contained in the hydrolysis extract, one part is taken and added with 5 mu L of 1 time thrombin solution, the mixture is quickly shaken up, and if the coagulation is carried out within 1min, the anticoagulant activity is less than 0.1 ATU; if the coagulation is not performed within 1min, the anticoagulation activity is larger than 0.1ATU, then 5 mu L of 1 time thrombin solution is dripped, the mixture is quickly shaken up, and if the coagulation is performed within 1min, the anticoagulation activity is smaller than 0.2 ATU; if the sample does not coagulate within 1min, the anticoagulation activity is more than 0.2ATU, 5 mu L of 1-fold thrombin solution is added dropwise, and the sample is shaken quickly until coagulation appears, so that the activity of the sample is known.
② one of the two solutions is dripped with 5 times of thrombin solution 5 mu L and shaken up rapidly, if the coagulation is not carried out within 1min, the anticoagulant activity contained in 50 mu L of hydrolysis extract is more than 0.5 ATU. Then dripping 20 times (400NIH/mL) of thrombin solution 5 μ L, shaking up rapidly, and if coagulation occurs within 1min, the anticoagulant activity is less than 2 ATU. At this time, a test tube is taken out from the water bath, 10 mu L of 5 times of thrombin solution is dripped, the test tube is shaken quickly, and if the test tube is solidified within 1min, the anticoagulant activity is less than 1 ATU. Dripping 5 μ L of 5 times of thrombin solution and 5 μ L of 1 time of thrombin solution on one branch, shaking rapidly, and if the solution coagulates within 1min, indicating that the anticoagulant activity is less than 0.6 ATU; if the blood sample is not coagulated within 1min, the anticoagulation activity is more than 0.6ATU, 5 mu L of 1-fold thrombin solution is dripped into the blood sample, and the blood sample is quickly shaken up, and if the blood sample is coagulated within 1min, the activity of the sample is less than 0.7 ATU. This is continued until the activity of the sample is detected.
Thirdly, detecting the activity of the sample, calculating the content and the specific activity
The activity is expressed in antithrombin activity units (ATU), and the assay is a thrombin titration improvement. The content is calculated according to the following formula:
U=20C
wherein C is the number of units containing ATU per 50. mu.L of the test solution (test sample or control sample), and U is the number of units containing ATU per 1mL of the test solution.
The results of the above tests on comparative examples 1 to 2 and examples 1 to 6 are shown in FIG. 1. Analysis in the figure shows that the anticoagulant activity of the samples prepared in the comparative example 1 and the sample prepared in the comparative example 2 have no obvious difference, which indicates that the anticoagulant efficacy of the artificially synthesized active peptide and the naturally active peptide has no great difference; the anticoagulant activity of the samples prepared in the embodiments 1-6 is more than 200TUA/mL, wherein the anticoagulant activity of the sample prepared in the embodiment 1 is 219.4TUA/mL, which is obviously higher than that of the sample prepared in the comparative example 1 and the sample prepared in the comparative example 2, and the anticoagulant activity of the polypeptide derivative prepared by chemically modifying the polypeptide is higher. The anticoagulant active peptide derivatives prepared in the embodiments 1-6 have high anticoagulant activity and have no obvious difference.
Test example 2:
in vitro thrombolytic Activity test
Materials: agarose, available from boao biology ltd, shanghai; CaCL2Solutions, purchased from Shanghai Sun Bioreagent, Inc.; plasma, purchased from Guangzhou Jianyang Bioreagent, Inc.; nattokinase, available from Nippon Bioreagent.
The experimental steps are as follows:
0.1g agarose is poured into 30mL distilled water, heated to boiling, cooled to 50 deg.C, and 5mL phosphate buffer solution of 0.05moL/L (pH 7.4) is added, 1.5mL plasma is added, and 0.025moL/L CaCL is added2Solution 1.5mL, stir rapidly until homogeneous, start the reaction and pour into a (d 9cm) petri dish while warm, spread evenly and have a thickness of about 1 cm. Spreading uniformly, standing horizontally for 30min, cooling and solidifying gel in the plate, perforating with a perforator with inner diameter of 0.45cm, injecting 1mg/mL sample solution and blank control solution 50 μ L, and placing in a constant temperature incubator at 37 deg.C. After 40h, the size of the lysis ring was observed, the diameter of the ring was recorded, the area of the ring was calculated, and the thrombolytic activity was expressed by the area of the ring. The area calculation formula of the dissolving ring is as follows:
area of dissolving ring (mm)2) [ (d major axis + d minor axis)/4]2X pi-0.159 (wherein 0.159 is the area of the hole)
The results of the above tests on the samples prepared in comparative example 2 and examples 1 to 6 are shown in FIG. 2. From the analysis in the figure, comparative examples 1 and 2 were preparedThe area of the sample has no obvious difference, which shows that the thrombolytic effect of the artificially synthesized active peptide and the natural active peptide has no great difference; the sample prepared in example 1 had a loop area of 390.4mm2The expression is obviously higher than that of the comparative example 1 and the comparative example 2, and shows that the polypeptide derivative prepared by chemically modifying the polypeptide has higher thrombolytic activity. The anticoagulant active peptide derivatives prepared in the embodiments 1-6 have high anticoagulant activity and have no significant difference. The active peptide derivative prepared by the invention can be directly orally taken, and the human digestive juice has little influence on the activity after the active peptide derivative is orally taken.
Test example 3:
half-life determination
1. Linear range
The sample is dissolved in 5mmoL/L ammonium acetate solution (containing 0.1% (v/v) formic acid), and 10 concentrations are set within the range of 1-1000 ng/L, namely 1, 5, 10, 20, 50, 100, 150, 250, 500, 1000 ng/microliter. Accurately measuring 1.0mL blank plasma in 10 test tubes, and adding corresponding sample standard substances according to the designed concentration. Processing the sample according to the process, performing RP-HPLC (reverse phase high performance liquid chromatography) determination, and performing linear regression according to the area under the curve of the sample standard substance and the concentration of the sample standard substance to obtain a regression equation;
2. blood concentration
And obtaining RP-HPLC curves of the samples in the plasma after different time, fitting the plasma concentration of the samples, making a curve graph to obtain a plasma concentration curve regression equation, and further calculating to obtain the half-life period.
The results of the above tests on comparative examples 1 to 2 and example 1 are shown in Table 1.
TABLE 1 four indices of blood coagulation after different times of sample testing
According to the data fitting calculation in the table, the half-life period of the comparative example 1 is 29 +/-2 min, the half-life period of the comparative example 2 is 48 +/-4 min, and the half-life period of the example 1 is 90 +/-3 min; the half-life period of the comparative example 2 is longer than that of the comparative example 2, which shows that the artificially synthesized anticoagulant active peptide has higher stability; the half-life period of the example 1 is obviously higher than that of the comparative example 2, which shows that the anticoagulant active peptide derivative prepared by chemical modification can effectively avoid the decomposition of in vivo enzyme and further improve the stability of the anticoagulant active peptide derivative.
Test example 4:
determination of antioxidant Activity
The antioxidant activity of the prepared samples was evaluated by DPPH free radical scavenging activity assay. Accurately sucking 2.00mL of sample solutions with concentrations of 0.1, 0.5, 1, 5, 10, and 20mg/mL respectively into test tubes, adding 2.00mL of 0.1M DPPH-ethanol solution respectively, mixing, reacting at room temperature in the dark for 30min, and measuring the light absorption value (A) of the solution at 517nm2). Meanwhile, positive control (glutathione, GSH) is set, and the light absorption value is A1Blank control (distilled water instead of sample) absorbance value is A0. DPPH clearance was calculated as follows:
DPPH clearance (%) [1- (a)2-A0)/A1]×100%
The test samples were the polypeptides and polypeptide derivatives prepared in comparative example 2 and examples 1 to 6, and the results are shown in FIG. 3. From the analysis in the figure, the DPPH & clearance rate of example 1 is 94.7%, which is obviously higher than that of comparative example 2, and the anticoagulant active peptide derivative prepared has better antioxidant activity. The samples prepared in examples 1-6 have no significant difference in antioxidant activity.
Conventional techniques in the above embodiments are known to those skilled in the art, and therefore, will not be described in detail herein.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Sequence listing
<110> Guangdong ocean university
<120> anticoagulant active peptide derivative, preparation method and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6
<212> PRT
<213> sea bream and preserved fish (Apogon aureus & Pseudomonas fulvidraco)
<400> 1
Phe Lys His Met Asn Glu
1 5
Claims (10)
1. An anticoagulant active peptide, the amino acid sequence of which is: Phe-Lys-His-Met-Asn-GLu.
2. An anticoagulant active peptide according to claim 1, wherein: the anticoagulant active peptide has anticoagulant activity and thrombolytic effect, wherein the anticoagulant activity is more than 150 ATU/mL.
3. The anticoagulant active peptide of claim 1, wherein the anticoagulant active peptide is used for preparing medicines for treating thrombotic cardiovascular and cerebrovascular diseases.
4. Use of the anticoagulant active peptides of claim 1 in the preparation of anticoagulant products.
5. The anticoagulant active peptide of claim 1, wherein the functional food is a powder, a tablet, an oral liquid or a capsule.
6. An anticoagulant active peptide derivative, the sequence of which is: X-Phe-Lys-His-Met-Asn-GLu, wherein the terminal group X is p-hydroxycinnamic acid, 2-amino-3- (2, 3-dihydroxyphenyl) propionic acid and 2-amino-3- (4-hydroxy-3-methoxyphenyl) propionic acid.
7. An anticoagulant active peptide derivative according to claim 6 wherein: in the sequence, Phe is in D configuration or L configuration, and the rest is in L configuration.
8. The anticoagulant active peptide derivative of claim 6, wherein the anticoagulant active peptide derivative is used for preparing medicines for treating thrombotic cardiovascular and cerebrovascular diseases.
9. Use of an anticoagulant active peptide derivative of claim 6 in the preparation of an anticoagulant product.
10. The anticoagulant active peptide derivative of claim 6, which is used for preparing functional food with anticoagulant and thrombolytic effects, wherein the functional food is powder, tablets, oral liquid or capsules.
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